An Inverted Repeat Triggers Cytosine Methylation of Identical Sequences in Arabidopsis  Bradley Luff, Laura Pawlowski, Judith Bender  Molecular Cell  Volume 3, Issue 4, Pages 505-511 (April 1999) DOI: 10.1016/S1097-2765(00)80478-5

Figure 1 PAI Gene Structure in Col and WS The MspI restriction maps of the WS PAI1–PAI4 locus, the Col PAI1 locus, and the PAI2 and PAI3 loci (which are identical between WS and Col) are shown. Sites internal to the PAI-identical sequences and the next closest flanking sites are indicated by M. Black arrows indicate PAI genes, with PAI3 hatched to represent reduced identity between this gene and its sister genes. Gray arrows indicate 2.9 kb direct repeat sequences that flank the WS PAI1–PAI4 genes and the Col PAI1 gene. The direct repeat downstream of Col PAI1 is partially deleted. The region covered by the PAI probe used in Southern blot analyses is indicated. The white arrowheads in the PAI4 gene and the pai4 5′ partial duplication indicate the orientation and extent of the duplicated sequences. The white box indicates a patch of heterologous sequence upstream of Col PAI1. Molecular Cell 1999 3, 505-511DOI: (10.1016/S1097-2765(00)80478-5)

Figure 2 Methylation Patterns in PAI Promoter Regions Bisulfite genomic sequencing of methylation patterns was performed for the top and bottom strands of the indicated PAI genes, with eight independent molecules sequenced for each strand. Vertical lines indicate the positions of cytosines, with the height of each line representing how many sequenced molecules had 5-Me-C at that position. Black indicates cytosines in the context 5′-CG-3′, blue indicates cytosines in the context 5′-CNG-3′, and red indicates cytosines in an asymmetric context. Asterisks indicate sites where none of the sequenced molecules had a 5-Me-C. The black horizontal line indicates the region of PAI identity, and the gray horizontal line indicates flanking upstream heterologous sequence unique to each gene. For each PAI gene, the transcription start site lies just interior to the point where PAI3 identity to PAI1 and PAI2 begins. Molecular Cell 1999 3, 505-511DOI: (10.1016/S1097-2765(00)80478-5)

Figure 3 De Novo Methylation in WS × Col Hybrids (A) F2 progeny of WS × Col crosses were screened for individuals homozygous for all permutations of the WS and Col PAI loci (7 Hyb1, 2 Hyb2, 4 Hyb3, 4 Hyb4, 4 Hyb5, and 1 Hyb6). DNA prepared from F3 progeny of individual hybrids was tested for PAI methylation by HpaII(H)/MspI(M) Southern blot with an internal PAI probe. (B) The same hybrid lines illustrated in (A) were inbred for three more generations, and DNA prepared from F6 progeny was tested for PAI methylation. Black indicates WS DNA, and gray indicates Col DNA. Arrows indicate PAI genes. The arrow corresponding to PAI3 is hatched to represent the reduced identity between this gene and its sister genes. Boxes around arrows indicate cytosine methylation, with the thickness of the line corresponding to the density of methylation observed. The upper chromosome corresponds to chromosome 1 carrying the PAI1–PAI4 locus and the unlinked PAI3 gene. The lower chromosome corresponds to chromosome 5 carrying the PAI2 gene. Asterisks indicate the positions of bands diagnostic of methylation. Molecular Cell 1999 3, 505-511DOI: (10.1016/S1097-2765(00)80478-5)

Figure 4 A Promoterless pai1–pai4 Inverted Repeat Transgene Becomes Methylated De Novo DNA was prepared from an inbred line carrying the homozygous pai1–pai4 transgene for two (2) or four (4) generations and analyzed by HpaII (H)/MspI (M) Southern blot with an internal PAI probe. Asterisks indicate the positions of bands diagnostic of methylation. The molecular weights of unmethylated and methylated transgene bands are indicated in the right margin. LB is the left border, RB is the right border, and kanR is the selectable marker on the transgene. Molecular Cell 1999 3, 505-511DOI: (10.1016/S1097-2765(00)80478-5)